Abstract In this application, we propose to apply a novel chemoenzymatic method developed by Prof. Lai-Xi Wang at University of Maryland (being licensed to GlycoT Therapeutics LLC) to construct a focused library of stable isotope-labeled Fc glycopeptides of IgG subclasses IgG1-4, which will be highly valuable as standards for absolute quantification of Fc glycosylation changes in human sera from patients and for quality control in producing IgG antibody-based therapeutics. Aberrant glycosylation is often associated with disease progressions and can serve as unique biomarkers. For example, Immunoglobulin G (IgG) is an important class of immunological glycoproteins. All IgGs contain an N-glycan at conserved Asn297 position in the Fc domain, and about 30% of IgGs also contain N-glycans at the Fab domains. The N-glycan linked to Asn297 dramatically modulates the activity of IgG, and aberrant change of Fc glycosylation also observed in many diseases. Thus, a robust and employable platform for site-specific quantitation of Fc glycosylation would be of tremendous value for monitoring biologics production (quality control) and for discovery and detection of specific biomarkers associated with various diseases. The use of MS in combination with isotopically enriched and quantified internal standards for quantitative analysis has become routine in metabolomics and proteomics. However, comprehensive stable isotope-labeled glycopeptide standards are lacking for site-specific quantitation of IgG Fc glycans. Although total chemical glycopeptide synthesis may serve this unmet need, it is still a formidable task, which requires highly specialized skills and is always tedious and time-consuming. In this application, we propose to apply a novel chemoenzymatic method to construct a focused library of stable isotope-labeled Fc glycopeptides of IgG subclasses IgG1-4. The chemoenzymatic technology exploits the transglycosylation activity of a class of endoglycosidases (ENGases) mutants that permits the ?native ligation? between free glycan and GlcNAc-tagged peptide to form homogeneous glycopeptide with native glycosidic linkage. The availability of these novel mutant enzymes, together with the highly convergent nature of the approach, enables a quick assembly of a library of diverse glycoforms of the IgG glycopeptides. We will perform two specific aims to achieve the goal. Specific Aim 1 is to establish a facile and convergent chemoenzymatic method for synthesizing stable isotope-labelled IgG-Fc glycopeptides; Specific Aim 2 is to expand the Fc glycopeptide library to diverse structures by enzymatic sugar chain trimming and extensions. We expect that a successful execution of the aims will lead to the construction of the designed stable istope labelled Fc glycopeptides ready for delivery for the research and development community for MS-based analysis and absolute quantitation of site-specific glycosylation of IgGs. .